JP2002173429A - Immunopotentiator for fishes and feed for cultured fish - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an immunopotentiator for fishes capable of increasing an immunization of cultured fish and controlling occurrence of fish diseases, and to provide a feed for cultured fish containing the immunopotentiator for fishes. SOLUTION: This immunopotentiator comprises 5'-nucleotide as an active ingredient. This feed for cultured fish has immunopotentiating effect. Consequently, the immunopotentiator increases immunization of cultured fish and has a controlling effect on fish diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to an immunopotentiator for fish containing 5'-nucleotide as an active ingredient.
The present invention relates to a feed for fish farming having an immunopotentiating effect containing a nucleotide as an active ingredient.

[0002]

2. Description of the Related Art In recent years, in the aquaculture industry, overcultivation of farmed fish tends to increase more and more for the purpose of improving management efficiency. In this kind of breeding situation, many cases have been reported in which once a disease develops, the entire case is infected in a short time and all fish are lost. Therefore, prevention of fish disease is an important issue directly linked to farmed fish management.

[0003] Therefore, as a countermeasure for preventing and after the onset of fish disease, a method of using drugs such as vaccines and antibiotics is used. However, there is a problem that the vaccine can only obtain an effect against a specific fish disease, and the emergence of resistant bacteria and contamination of the surrounding environment in antibiotics.

[0004] For the purpose of solving such a problem, various immunopotentiators for increasing the immunity (resistance to disease) of cultured fish to prevent fish disease have been marketed and used in actual aquaculture sites. .

[0005] For example, a Suehirotake mushroom culture (Japanese Unexamined Patent Publication No.
70919), lactoferrin (JP-A-07-48274) contained in mammalian milk, peptidoglycan that is a cell wall component of bacterial cells (JP-A-11-256664), and the like are known. However, the above-mentioned immunopotentiating agents are not sufficiently effective or increase the economic burden, and thus cannot be said to have sufficiently solved the problem.

On the other hand, nucleic acids (deoxyribonucleic acid, ribonucleic acid) and nucleic acid-related substances have attracted attention as new immune enhancing substances. For example, a method for use in human dietary supplements (JP-A-61-195651) and a method for incorporating a nucleic acid-related substance into a formula for newborn and premature infants (JP-A-10-327804) are disclosed. Also, a method of mixing 5'-nucleotides with livestock feed for the purpose of suppressing diarrhea (Japanese Patent Publication No. 43-14388) has been disclosed.

However, the utilization of these nucleic acids and nucleic acid degradants as immunopotentiators is limited to mammals including humans. Nucleic acids and nucleic acid degradants are never used as fish immunopotentiators.

[0008]

An object of the present invention is to provide an inexpensive immunopotentiator which exerts a sufficient effect on fish.

[0009]

Means for Solving the Problems As a result of repeated studies to solve the above-mentioned problems, the present inventors have found that, among nucleic acids and nucleic acid-related substances whose effects have been limited to mammals, 5'-nucleotides have been replaced by fish. The present inventors have found an effect of enhancing immunity, and have completed the present invention. That is, administration of 5'-nucleotides can enhance the immunity of fish.

[0010] Further, since the fish immunopotentiator exerts a sufficient effect by oral administration, the present invention can also provide a feed for fish farming containing the fish immunopotentiator.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail.

The 5'-nucleotide used in the present invention is composed of a sugar, a purine or pyrimidine base, an ester-linked phosphate group, and a phosphate linked to the 5'-position. The constituent sugar is ribose or deoxyribose. Nucleotide is a name that includes substances such as 5′-thymidylic acid, 5′-uridylic acid, 5′-adenylic acid, 5′-guanylic acid, 5′-cytidylic acid, and 5′-inosinic acid. The 5'-nucleotide described above is a mixture of these various nucleotides at any ratio.

The immunopotentiator of the present invention may be in the form of a mixture with other immunopotentiators or antibiotics, or in the form of a mixture with production auxiliaries. It is desirable to contain 5% or more of 5'-nucleotide.

The immunopotentiator of the present invention can be used in combination with other immunopotentiators or antibiotics, thereby enhancing the effects of other immunopotentiators or antibiotics, or enhancing other immunopotentiators. It can also be expected to reduce the use of drugs or antibiotics.

The 5'-nucleotide used in the present invention is
It can be easily obtained by hydrolyzing a commercially available ribonucleic acid with 5'-phosphodiesterase. Ribonucleic acid is
It can be obtained by extraction and purification from yeast, bacteria and the like used for food and feed, but any source can be used, and the source is not limited.

In addition, it can be obtained not only by a hydrolysis method but also by a direct fermentation method using bacteria, and is not limited to a production method.

In addition, even for deoxyribonucleic acids having different constituent sugars, 5'-nucleotides can be easily obtained from commercially available deoxyribonucleic acids by hydrolysis with 5'-phosphodiesterase having deoxyribonucleic acid decomposing activity. Deoxyribonucleic acid can be obtained industrially by extracting and purifying from milt such as salmon and trout.However, any source of deoxyribonucleic acid can be used as a raw material for the product of the present invention. It is not limited to milt.

The feed for fish farming having an immunopotentiating effect containing 5'-nucleotide as an active ingredient in the present invention can be obtained by blending 5'-nucleotide with a commercially available compound feed. The addition rate is 0.01 to 10% by weight.
Is preferred. If the 5'-nucleotide addition rate is less than 0.01% by weight, no immunopotentiating effect is observed, and 10% by weight.
If the amount exceeds the limit, the effect of addition is saturated and the nutritional value of the feed for fish farming is impaired.

[0019] The fish culture that is the object of the present invention is carp,
Ayu, rainbow trout, eel, hamachi, Thailand, flounder and the like. The present invention exhibits an immunopotentiating effect on these fish farming from the stage of fry to raw fish.

[0020]

The present invention will be described in detail with reference to the following examples.

[Preparation Example 1 Preparation of 5'-nucleotide]
After preparing a 5% by weight solution of a commercially available RNA, 1% by weight of ribonuclease (manufactured by Amano Pharmaceutical Co., Ltd.) was added to the RNA, reacted at pH 5, 70 ° C. for 10 hours, and spray-dried to 5 ′.
-Nucleotides were obtained. Various nucleotide compositions of the obtained 5′-nucleotide were determined by analysis using HPLC. Table 1 shows the analysis results. In addition, the 5'-nucleotide purity of this product was 88.0%.

[0022]

[Table 1] (Table 1)

[Preparation Example 2 Preparation of 5'-nucleotide]
After preparing a 5% by weight solution of a commercially available DNA, 1% by weight of ribonuclease (manufactured by Amano Pharmaceutical Co., Ltd.) was added to the DNA, reacted at pH 5, 70 ° C. for 10 hours, and spray-dried to 5 ′.
-Nucleotides were obtained. Various nucleotide compositions of the obtained 5′-nucleotide were determined by analysis using HPLC. Table 2 shows the analysis results. In addition, the 5'-nucleotide purity of this product was 91.0%.

[0024]

[Table 2]

[Example 1] (Measurement of phagocytic ability of phagocytes) Purchased from a commercial fish farm,
Using 5 carp fish (average body weight: 150 g) each, 5′-nucleotide prepared by the method of Production Example 1 was dissolved in physiological saline, and forcibly administered orally using a sonde. The dose is 0.
The doses were 075 mg, 0.75 mg, and 7.5 mg (each dose per fish). The control group received the same amount of physiological saline alone. One day later, leukocytes were separated from carp kidney and adjusted to a certain number. After mixing with a latex bead suspension and reacting for 2 hours, the number of leukocytes incorporating the latex beads was counted. Table 3 shows the results.

[0026]

[Table 3]

In all the groups to which the 5'-nucleotide was administered,
A significant difference from the control group was recognized. Therefore, the effect of increasing the phagocytic ability of carp leukocytes was recognized by 5'-nucleotide.

[Example 2] (Measurement of bactericidal action of phagocytic cells) Production Example 1 using 5 carp (average body weight: 150 g) each purchased from a commercial fish farm
The 5′-nucleotide prepared by the method described above was dissolved in physiological saline and forcibly administered orally using a sonde. The dose was 0.075 mg, 0.75 mg and 7.5 mg (each dose per fish). The control group received the same amount of physiological saline alone. One day later, leukocytes were separated from carp kidney and attached to a 96-well microplate.
A was added and stimulated, and the generation of hydrogen peroxide involved in the bactericidal action in the phagocytic cells was measured by the color change (absorbance at 620 nm) of the NBT reagent. Table 4 shows the results.

[0029]

[Table 4]

In the group to which 7.5 mg of 5'-nucleotide / tail was administered, a significant difference from the control group was observed. Therefore,
The effect of increasing the bactericidal action of carp leukocytes was observed with 5'-nucleotide.

Example 3 (Measurement of lysozyme activity) Purchased from a commercial fish farm,
Using 5 carp (average body weight: 150 g) each, 5′-nucleotide prepared by the method of Production Example 2 was dissolved in physiological saline, and forcibly administered orally using a sonde. The dose is 7.
The dose was 5 mg (dose per fish). The control group received the same amount of physiological saline alone. One day later, serum was collected, mixed with a heat-killed suspension of Micrococcus lysodeikticus, reacted at 45 ° C. for 30 minutes, and the turbidity of the test solution was measured to measure the lysozyme activity of the serum. The unit per 0.01 of the reduced turbidity was defined as 1 unit. Table 5 shows the results.
Shown in

[0032]

[Table 5]

A significant difference was observed between the group to which 5′-nucleotide was administered and the control group. Therefore, the effect of increasing the lysozyme activity in carp serum was recognized by 5′-nucleotide.

Example 4 (Measurement of Complement Activity) Using 5 carp (average body weight: 150 g) each purchased from a commercial fish farm, 5′-nucleotide prepared by the method of Production Example 2 was physiological saline. It was dissolved in water and forcibly administered orally using a sonde. The dose is 7.5m
g (dose per fish). The control group received the same amount of physiological saline alone. One day later, serum was collected,
Complement activity was measured as follows. 10 mM MgCl
₂ and 10 mM EGTA (ethyleneglycol-bis tetraacetic
Rabbit erythrocytes were mixed with serum serially diluted with 0.1% gelatin-veronal buffer containing acid), reacted at 45 ° C. for 30 minutes, and the degree of hemolysis in each system was measured with an absorbance meter. The dilution factor of the serum at which the rabbit erythrocytes were hemolyzed 50% was defined as the complement value (ACH50). Table 6 shows the results.

[0035]

[Table 6]

There was a significant difference between the group to which 5'-nucleotide was administered and the control group. Therefore, the effect of increasing complement activity in carp serum was recognized by 5′-nucleotide.

Example 5 (Test for Infection of Fish Disease Bacteria) Carp (average body weight: 150 g) purchased from a commercial fish farm was used in each of 12 fishes, and a 5'-nucleotide dissolved in physiological saline was used as a probe. Orally. The dose was 7.5 mg (dose per fish). The control group received the same amount of physiological saline alone. One day later, the fish disease bacterium Aeromonas hydrop
hia was inoculated with 3 × 10 ⁷ CFU per fish, and the changes in the number of bacteria in the blood, kidney and liver were collected and mixed at 2, 4, 8, and 12 hours after infection. It was measured by counting the number of bacteria. Tables 7 to 9 show the results.

[0038]

[Table 7]

[0039]

[Table 8]

[0040]

[Table 9]

At 12 hours after carp inoculation with the fish disease bacteria, the numbers of bacteria in blood, kidney and liver were all lower in the group to which 5'-nucleotide was administered than in the control group. Therefore, it was confirmed that 5'-nucleotide had an effect of increasing the action of removing fish disease bacteria in carp.

Example 6 (Preparation of fish feed containing 5'-nucleotide) The 5'-nucleotide of Production Example 1 and 5'-nucleotide of Production Example 2 were added to the fish feed shown in Table 10. The feeds A and B for fish farming were prepared by mixing at a ratio of 5% by weight.

[0043]

[Table 10]

Example 7 Phagocytic Activity of Carp Fed with Fish Feed Containing 5'-Nucleotide After feeding fish feeds A and B of Example 6 for 5 days (10 Tail), the phagocytic activity of carp was measured by the method of Example 1. The control is shown in Table 10
The feed for fish farming shown in Table 2 was given. Table 11 shows the results.

[0045]

[Table 11]

The group fed with fish feeds A and B to which 5'-nucleotide was added showed a significant difference from the control group. Therefore, the effect of increasing the phagocytic ability of carp leukocytes was found in a fish feed to which 5′-nucleotide was added.

[Example 8] (Lysozyme activity of carp fed fish feed containing 5'-nucleotide) The fish feeds A and B of Example 6
After feeding to carp for 10 days (10 fish in each section), lysozyme activity of carp was measured by the method of Example 3. The control is
The fish feeds shown in Table 10 were given. Table 12 shows the results.

[0048]

[Table 12]

The group fed with fish feeds A and B to which 5'-nucleotide was added showed a significant difference from the control group. Therefore, the effect of increasing the lysozyme activity of carp was found in the fish feed to which 5′-nucleotide was added.

[0050]

EFFECT OF THE INVENTION The fish immunity enhancer comprising 5'-nucleotide as an active ingredient and the feed for fish farming having 5'-nucleotide as an active ingredient provided by the present invention are effective in immunizing fish cultivation. And can effectively suppress the occurrence of fish disease, which is extremely useful.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07H 19/20 C07H 19/20 (72) Inventor Katsuhiro Makoto 1-12-1 Yurakucho, Chiyoda-ku, Tokyo Japan Inside Paper Manufacturing Co., Ltd. EA16 MA01 MA04 NA14 ZB09 ZC65

Claims (2)

[Claims] 1. An immunopotentiator for fish comprising a 5′-nucleotide as an active ingredient. 2. A feed for fish farming, comprising the immunopotentiator according to claim 1.